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Establecimiento in Vitro De Taxus Globosa Schltdl

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Establecimiento in Vitro De Taxus Globosa Schltdl Establecimiento in vitro de Taxus globosa Schltdl. UNIVERSIDAD DE GUADALAJARA CENTRO UNIVERSITARIO DE CIENCIAS BIOLÓGICAS Y AGROPECUARIAS “Establecimiento in vitro de Taxus globosa Schltdl.” TRABAJO DE TITULACIÓN MODALIDAD TESIS QUE PARA OBTENER EL TÍTULO DE LICENCIADO EN BIOLOGÍA PRESENTA: C. JOSÉ LUIS PAZ ROBLES DIRECTORA: M. EN C. MARIBEL M. LOERA QUEZADA ASESOR: DR. CARLOS RAMÍREZ SERRANO LAS AGUJAS, ZAPOPAN, JAL., SEPTIEMBRE DEL 2015 - 1 - Establecimiento in vitro de Taxus globosa Schltdl. - 2 - Establecimiento in vitro de Taxus globosa Schltdl. - 3 - Establecimiento in vitro de Taxus globosa Schltdl. Este trabajo formó parte del proyecto: “Desarrollo de un Protocolo para la Micropropagación de Taxus globosa Schltdl.” Convocatoria CONAFOR- CONACYT-2004-C04-06, financiado por el Comité Técnico y de Administración del Fondo Sectorial para la Investigación, el Desarrollo y la Innovación Tecnológica Forestal. El presente trabajo se realizó en el Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), en el Laboratorio de Biotecnología Vegetal. Dirección: Av. Normalistas 800, Colinas de La Normal, 44270 Guadalajara, Jal. Teléfono: 01 33 3345 5200 - 4 - Establecimiento in vitro de Taxus globosa Schltdl. AGRADECIMIENTOS Al Universo por otorgarme a estos seres maravillosos…..Efraín Paz y Francisca Robles mis Padres….. con quienes comparto parte de sus alelos, siendo ellos autores de lo que soy…..gracias por su soplo de vida, su amor y la trasmisión de sus valores…..los amos. A mi cómplice de aventuras, viajes…camarada de partido, de ideas… ese ser autentico y único que me ha acompañado en lo largo de mi vida…..Mara Paz mi Hermana…..gracias por todo y por darle luz a mi existir. A mis otros ocho hermanos genéticos: Efraín, Adolfo, Claudia, Ricardo, Gerardo, Susana, Rubén y Raúl por ser pilares y formar parte de mi vida, a la cual le han añadido sabor y momentos felices. A mis hermanos espirituales: Olivia Distancia, Ana María Gaspar, Xochitlquetzalli Estrada, Tere Sahagún y Juan P.R. Arana por coincidir en mi camino otorgándole gracia y armonía…los quiero. A los que se me adelantaron en mi camino transformándose en rayos de luz: Ricardo Sainz, Jesús Uribe y a ti Ángel Zúñiga Saavedra por haber estado en los momentos más cruciales de mi existir y darle serenidad a mi vida…siempre estarán presentes en mí… A esos seres que le otorgaron ese toque de investigación y haberme forjado perspectivas en mi formación: Dr. Carlos Ramírez Serrano y M. en C. Maribel Loera Quezada, gracias por haber aportado enriquecido mi conocimiento…. A esos grandes personas que intervinieron y aportaron conocimiento: Dr, Armando Arias, Dra, Lucila Méndez y M.C. Rafael Soltero, gracias a ustedes mi trabajo de investigación fue tomando forma hasta su materialización…gracias. Con Amor a todos Ustedes…….. - 5 - Establecimiento in vitro de Taxus globosa Schltdl. INDICE GENERAL ÍNDICE DE TABLAS...............................................................................................iii ÍNDICE DE FIGURAS……………………………………………………………………iv ABREVIATURAS……………...................................................................................v RESUMEN...............................................................................................................vi 1. INTRODUCCIÓN...........................................................................................1 1.1 Antecedentes generales…………………………………………………….2 1.1.1 Género Taxus…………………………………………………………..2 1.1.2 Descripción botánica del género Taxus……………………………..4 1.1.3 Utilización e importancia económica del género Taxus…………...5 1.1.4 Taxus globosa Schltdl…………………………………………………7 1.1.5 Historia del paclitaxel………………………………………………...11 1.1.6 Problemática de abastecimiento y fuentes de paclitaxel ………13 1.2 Establecimiento in vitro…………………………………………………….15 1.2.1 Selección del material vegetal………………………………………15 1.2.2 Desinfección del material vegetal…………………………………..17 1.2.3 Condiciones del cultivo e incubación………………………………18 1.2.4 Contaminación in vitro……………………………………………….19 1.2.5 Oxidación in vitro …………………………………………………….20 1.3 Micropropagación…………………………………………………………..23 1.3.1 Proliferación de yemas axilares…………………………………….24 1.3.2 Organogénesis e embriogénesis somática………………………..25 1.3.3 Importancia económica de la micropropagación………………….25 1.3.4 Proliferación de yemas axilares en el género Taxus…………..…26 1.3.5 Organogénesis y embriogénesis somática en el género Taxus...28 1.4 Metabolitos secundarios y cultivo de líneas celulares en suspensión..28 1.4.1 Metabolitos secundarios……………………………………………..28 1.4.2 Cultivo de líneas celulares en suspensión………………………...30 1.4.3 Importancia del cultivo de líneas celulares en suspensión………31 1.4.4 Cultivo de callos en el género Taxus………………………………32 1.4.5 Cultivo de líneas celulares en suspensión en el género Taxus ...35 2. PLANTAMIENTO DEL PROBLEMA………………………………………….37 3. JUSTIFICACIÓN………………………………………………………………...37 - 6 - Establecimiento in vitro de Taxus globosa Schltdl. 4. HIPÓTESIS………………………………………………………………………38 5. OBJETIVO GENERAL………………………………………………………….38 5.1 Objetivos específicos………………………………………………………38 6. METODOLOGÍA………………………………………………………………...39 6.1 Fase 1: Establecimiento in vitro de Taxus globosa Schltdl……………39 6.1.1 Material vegetal……………………………………………………….39 6.1.2 Desinfección de los explantes………………………………………39 6.1.3 Medio y condiciones de cultivo……………………………………..41 6.2 Fase 2: Inducción de yemas axilares de Taxus globosa Schltdl……...42 6.2.1 Material vegetal………………………………………………………42 6.2.2 Medio y condiciones de cultivo……………………………………..42 6.3 Fase 3: Inducción de callo en acículas de Taxus globosa Schltdl……43 6.3.1 Material vegetal………………………………………………………43 6.3.2 Medio y condiciones de cultivo para la inducción de callo………43 6.4 Fase 4: Iniciación de la organogénesis indirecta de T. globosa Schltdl 6.4.1 Material vegetal……………………………………………………….43 6.4.2 Medio y condiciones de cultivo para la inducción de brotes adventicios…………………………………………………………….43 6.5 Fase 5: Iniciación del cultivo de líneas celulares en suspensión de Taxus globosa Schltdl……………………………………………………..46 6.5.1 Material vegetal……………………………………………………….46 6.5.2 Medio y condiciones de cultivo para el inicio de líneas celulares en suspensión……………………………………………………….. 46 7. RESULTADOS Y DISCUSIONES...............................................................47 7.1.1 Desinfección…………………………….………………………………..47 7.1.2 Oxidación…………………………….……………………………………51 7.3 Inducción de las yemas axilares…………….………………………….57 7.4 Inducción de callo………………………………………........................61 7.5 Inducción de brotes adventicios………………………………………..66 7.6 Iniciación del cultivo de líneas celulares en suspensión…………….68 8. CONCLUSIONES……………………………………………………………….71 9. PERSPECTIVAS………………………………………………………………..73 10. ANEXOS…………………………………………………………………………74 BIBLIOGRAFÍA……………………………………………………………………..75 - 7 - Establecimiento in vitro de Taxus globosa Schltdl. ÍNDICE DE TABLAS Tabla 1. Especies del género Taxus y su ubicación geográfica……..…...............3 Tabla 2. Clasificación taxonómica de la especie Taxus globosa Schltdl…..……..7 Tabla 3. Eventos históricos del paclitaxel……………………………………..……12 Tabla 4. Diferentes alternativas para la obtención del paclitaxel y alternativas en el cultivo de tejidos vegetales in vitro……………….……………..….14 Tabla 5. Trabajos in vitro en el género Taxus en donde se presentó el proceso de oxidación y su solución………………………………………22 Tabla 6. Proliferación de yemas axilares en el género Taxus…….……………...27 Tabla 7. Propagación in vitro vía organogénesis y embriogénesis somática en el género Taxus………………………………………………………. ..29 Tabla 8. Trabajos in vitro en la inducción de callo en varias especies del género Taxus………………………………………………………………. 32 Tabla 9. Producción de paclitaxel por cultivo de líneas celulares en suspensión en diversas especies de Taxus….………………………… 35 Tabla 10. Nomenclatura de los explantes de los árboles de Taxus globosa…... .39 Tabla 11. Tratamientos utilizados con sus respectivas concentraciones de reguladores de crecimiento para la inducción de yemas axilares…… 43 Tabla 12. Tratamientos con sus respectivas concentraciones de reguladores de crecimiento para la inducción de callo………………………………. 44 Tabla 13. Tratamientos con sus respectivas concentraciones de reguladores crecimiento para la inducción de brotes adventicios………………….. 45 Tabla 14. Porcentajes de contaminación de los grupos controles y experimentales de los 10 árboles utilizados……………………………. 49 Tabla 15. Porcentajes de oxidación de los grupos controles y experimentales de los 10 árboles utilizados.……………………………………………… 52 Tabla 16. Valores determinados por el ANOVA para No Brotes, Tamaño, Color y Apariencia en explantes con meristemo apical….…………… 57 Tabla 17. Valores determinados por la prueba de rangos múltiples (LSD) por tratamiento para el número de brotes en explantes con meristemo apical y decapitados……………………..………….…………………….. 58 Tabla 18. Valores determinados por el ANOVA en los explantes con meristemo apical para No Brotes, Tamaño, Color y Apariencia, en la interacción de las citocininas KIN y 2iP…………………………. ..59 Tabla 19. Valores determinados por la prueba de rangos múltiples (LSD) en el efecto de la concentración de 2iP para el número de brotes en explantes con meristemo apical…………………………………………. 59 Tabla 20. Valores determinados por el ANOVA en los explantes decapitados para No Brotes, Tamaño, Color y Apariencia, en la interacción de las citocinina KIN y 2iP……………………………………………………. 60 Tabla 21. Valores determinados por la prueba de rangos múltiples (LSD) en el efecto de la concentración
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